产单核细胞李斯特菌的PFGE分子分型分析

对50株不同来源的产单核细胞李斯特菌（Lm）进行PFGE分型,比较食源、环境、人源菌株之间的相似性。结果显示,同一市场内,环境污水分离株和新鲜食品分离株表现出较高同源性,新鲜食品分离株与即食性（RTE）食品分离株相似性很高,临床脑膜炎病人的1株Lm与RTE食品分离株在进化上显示出了很近的亲缘关系。50株Lm的分子分型分析提示了菌株从市场环境污水-新鲜食品-RTE食品-人的传播模式,为食源性疾病溯源追踪提供了新的思路。     

鸡沙门氏菌脉冲场凝胶电泳分型研究

为应用脉冲场凝胶电泳(PFGE)从分子水平上对禽源沙门氏菌之间的差别进行分析和研究,本研究采用自动微生物鉴定仪对12株疑似鼠伤寒沙门氏菌和15株疑似鸡白痢沙门氏菌进行了鉴定.以限制性核酸内切酶Xba I对其全基因组DNA进行酶切、PFGE分型,Info Quest FP聚类分析软件对电泳结果进行分析.结果显示,共鉴定出11株鼠伤寒沙门氏菌、1株圣保罗沙门氏菌、6株阿姆斯特丹沙门氏菌、5株亚拉巴马沙门氏菌、1株鸡白痢沙门氏菌、2株纽波特沙门氏菌和2株肠炎沙门氏菌,一共分为12个PFGE型.实验结果表明,江苏地区存在有不常见的沙门氏菌血清型,同种血清型之间的基因型差别较小(相似值为0.85～1),不同血清型之间差别较大(相似值为0.45～0.70).PFGE能从分子水平上对禽源沙门氏菌的基因组DNA进行分型.     

Comparison of pulsed-field gel electrophoresis with random amplified polymorphic DNA for typing of Mycoplasma synoviae

Pulsed-field gel electrophoresis (PFGE) analysis was developed and compared with random amplified polymorphic DNA (RAPD) method to type 18 Mycoplasma synoviae (MS) strains. All analysed strains were typeable by RAPD but only 89% of MS strains were typeable by PFGE because of DNA degradation. The discriminatory power of RAPD was greater than that of PFGE but the two techniques had a discriminatory index superior to 0.95, the threshold value for interpreting typing results with confidence. The in vitro, in ovo and in vivo reproducibility of both typing techniques was 100%. However, the interpretation of RAPD patterns was complicated because of inconsistent band intensity. Thus, these molecular typing techniques should be helpful for epidemiological studies of avian mycoplasma infections.     

Chromosome-sized DNA of Malassezia pachydermatis by pulsed-field gel electrophoresis.

The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes by using pulsed-field gel electrophoresis and their molecular sizes were calculated as 820, 1,100, 1,400, 1,470, 1,660 and 1,820 Kb, respectively. Comparison of electrophoretic patterns suggested that the chromosomes of M. pachydermatis were homozygous.     

Demonstration of polymorphism among Brucella ovis field isolates by pulsed-field gel electrophoresis

Brucella ovis is recognized worldwide as an important pathogen of sheep, and has also been identified in farmed deer in New Zealand. Previously, only one strain type of B. ovis has been identified. The objective of this paper was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. ovis to determine whether strain variations exist, whether sheep and deer are affected by the same strains, and to compare the performance of the rare-cutting restriction enzymes XbaI and SwaI. Ten B. ovis isolates from sheep and two from deer in New Zealand, as well as the type strain, were subjected to PFGE analysis using both XbaI and SwaI. PFGE of XbaI restriction fragments produced two banding patterns consisting of 27-28 bands, which were found to be 98% similar by cluster analysis, and were named X1 and X1a. PFGE of SwaI restriction fragments resulted in three banding patterns consisting of 13-15 bands each. Ten of the isolates had identical banding patterns and were named S1. One isolate differed by one band, representing a subtype named S1a. Two isolates differed by six bands, representing a different strain type of B. ovis and this was named S2. Cluster analysis showed S2 to be 78% similar to the S1/S1a cluster. Both strain types were isolated from both sheep and deer. Thus, two distinct strain types of B. ovis were identified in New Zealand, which is the first report of more than one strain type being identified worldwide. Neither strain was species-specific for sheep or deer. The restriction endonuclease SwaI was found to be more discriminatory than the enzyme XbaI, which has been used in previous studies.     

Epidemiological analysis of Salmonella enteritidis from human outbreaks by pulsed-field gel electrophoresis.

To determine the extent of genetic diversity among isolates of Salmonella enteritidis obtained from outbreaks in Fukuoka Prefecture, Japan, from 1989 to 1994, we analyzed a total of 55 isolates of S. enteritidis obtained from 13 distinct outbreaks with pulsed-field gel electrophoresis. These isolates showed three different patterns in pulsed-field profile with Bln I digestion. The groups A, B and C consisted of three outbreaks isolates (Dice coefficient, F = 1), of seven outbreaks (F = 0.56-0.94) and of three outbreaks (F = 0.65-0.78), respectively. This result suggests that a few limited clonal lines of S. enteritidis were successively causing outbreaks in this area from 1989 to 1994.     

Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis

Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P. aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm outbreaks of haemorrhagic pneumonia. Isolates from mink were separated into 34 distinct clones by PFGE subtyping. All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak. P. aeruginosa of specific PFGE types were found to cause clusters of outbreaks on several farms within a few weeks of each other. However, PFGE types of strains causing clusters of farm outbreaks changed from year to year. These results suggest that some outbreaks of haemorrhagic pneumonia are caused by pathogenic strains of P. aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment.     

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

Objectives To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis .
Design Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates.
Procedure DNAs from 35 Australian isolates of M para-tuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared.
Results The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales.
Conclusion Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis , and could be used to study the transmission of strains in Australia.     

使用Reveal试剂盒快速检测单增李斯特氏菌的研究

单核细胞增生李斯特氏菌(Listeria monocytogens,LM,简称为单增李斯特氏菌)是食品卫生上重要的病原菌。它广泛存在于自然界,可导致人和动物脑膜炎、流产、败血症等,病死率高达30%-70%,是目前人类最重要的食物源性病原菌之一。由于该菌生长温度范围较广,在     

Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis

Three hundred and forty-two Streptococcus uberis isolates were cultured from milk samples from subclinical and clinical cases of dairy cattle mastitis. The samples were collected from 15 different New Zealand farming regions, including eight specific farms, during field research trials and veterinary diagnostic investigations. Pulsed-field gel electrophoresis was used to determine and compare the degree of genetic dissimilarity between the restriction endonuclease fragment pattern of the 342 New Zealand and a single United States S. uberis isolate. The 343 isolates exhibited 330 different restriction endonuclease fragment patterns. The United States isolate had a pattern unlike any of the New Zealand isolates. Most of the isolates were genetically different strains (pattern deferred by at least 33%), but identical patterns were noted within the same or different quarters of an individual cow, different cows within the same farm, and from different cows from the same or different districts, farming regions or islands. Seven of the eight selected farms had at most only one pair of isolates with banding patterns, which differed by less than 33%. A high degree of dissimilarity was noted in individual herds in which all the samples were collected on the same day or over a 2-year period. The high degree of dissimilar isolates is an indication that S. uberis infections in New Zealand dairy cattle are largely due to the opportunistic nature of the organism in the cows' environment. Prevention and treatment of S. uberis mastitis will therefore need to be directed at a multitude of different strains present throughout the country as well as in individual herds.     

Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens

The aim of this study was to analyse the genetic diversity among Clostridium perfringens isolates from Danish broiler chickens since both sick and presumably healthy animals were investigated. Isolates (n=279) collected from chickens from 25 farms were analysed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI. A high genetic diversity was found. Isolates with different PFGE types were toxin typed by PCR and all were found to be of type A. The results showed that healthy broiler chickens carried several different C. perfringens clones both within a flock and even within individual birds, whereas flocks suffering from necrotic enteritis (NE) or cholangio-hepatitis carried only one or two clones.     

Genetic diversity of Mycobacterium avium subspecies paratuberculosis isolates from goats detected by pulsed-field gel electrophoresis

Paratuberculosis in goats occurs worldwide causing considerable economic losses mainly due to reduced milk production. Nowadays, there is still relatively little knowledge about the epidemiology of this disease in goats, and only a few epidemiological studies have been carried out in goats naturally infected with Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis). The objective of this study was to characterize forty four clinical caprine isolates of M. a. paratuberculosis by different molecular techniques (pulsed-field gel electrophoresis [PFGE], restriction fragment length polymorphism analysis coupled with hybridization to IS900, and IS1311 polymerase chain reaction-restriction enzyme analysis) to determine the most useful technique for molecular typing of caprine isolates, as well as to disclose the genetic variation amongst caprine isolates and the relationship with strains isolated from other animal species. PFGE was found to be the most discriminative technique identifying a total of 13 'multiplex' PFGE profiles, ten of which were novel profiles found only in caprine isolates to date. All isolates were genotyped as Type II strains, except two isolates that resembled the intermediate group referred as Type III.     

奶牛乳房炎金黄色葡萄球菌脉冲场凝胶电泳分型研究

为探讨新疆北疆奶牛乳房炎致病菌的流行规律,本研究采用Sma Ⅰ酶切,脉冲场凝胶电泳(PFGE)分型的方法对43株分离自新疆6个规模奶牛场隐性乳房炎奶样的金黄色葡萄球菌进行了分子分型比较研究.结果表明,所有菌株都能被PFGE法分型,43株金黄色葡萄球菌可分为A、B、C、D和E 5个基因型.A型株(22株,51.2%)有13个亚型,相似度在81.8%～96.8%之间,在5个奶牛场均分离到,是主要的流行株;B型(25.6%)、C型(14.0%)、D型(7.0%)各型别内菌株间的相似度为100%,E型仅有1株.不同地区主要流行株有差别:乌鲁木齐主要流行A型菌株,昌吉以A型和B型菌株为主,而奎屯主要流行C型和B型.有2个奶牛场流行株只有1个基因型,也有2种基因型(3个牛场)或3种基因型(1个牛场)同时存在,但以1种为优势株.这些结果提示,不同地区主要流行株有差别,多数奶牛场以1种流行株感染为主,不同牛场可能流行相同的菌株,在较大地域范围内某些流行株具有侵染优势.     

Evaluation of a 23S rDNA polymerase chain reaction assay for identification of Serpulina intermedia, and strain typing using pulsed-field gel electrophoresis

A polymerase chain reaction assay, amplifying a 1027 base pair portion of the 23S rDNA gene, was evaluated for identification of the intestinal spirochaete Serpulina intermedia. A total of 34 strains of S. intermedia isolated from pigs and chickens and 195 strains of other related species were tested. The optimised assay correctly identified all the S. intermedia strains, but generated 11 false positive reactions, giving a test sensitivity of 100% and a test specificity of 94.3%. The false positive reactions were generated from strains of four different species of intestinal spirochaetes, and the product was of the original predicted size. This suggests that the primer sites selected on the 23S rRNA gene were not completely specific for S. intermedia. Pulsed-field gel electrophoresis was then developed to investigate diversity amongst the S. intermedia strains. All strains tested had distinct DNA banding patterns using Mlu1, although three isolates from chickens on the same farm appeared closely related. The collection exhibited considerable genetic diversity, and strains from pigs and chickens were distributed in clusters throughout the dendrogram produced. The most closely related porcine and avian strains shared only 62% similarity.     

The diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field gel electrophoresis

Over the years, pulsed-field gel electrophoresis (PFGE) has been proven a robust technique to type isolates with a high resolution and a good reproducibility. In this study, a PFGE protocol is described for the typing of Mycoplasma hyopneumoniae isolates. The potential of this technique was demonstrated by comparing M. hyopneumoniae isolates obtained from the same as well as from different herds. The use of two different restriction enzymes, SalI and ApaI, was evaluated. For each enzyme, the resulting restriction profiles were clustered using the unweighted pair group method with arithmetic means (UPGMA). For both obtained dendrograms, the included isolates of the related M. flocculare species clustered separately from all M. hyopneumoniae isolates, forming the root of the dendrograms. The PFGE patterns of the M. hyopneumoniae isolates of different herds were highly diverse and clustered differently in both dendrograms, illustrated by a Pearson's correlation coefficient of only 0.33. A much higher similarity was observed with isolates originating from different pigs of a same herd. The PFGE patterns of these isolates always clustered according to their herd and this for both dendrograms. In conclusion, the results indicate a closer relationship of M. hyopneumoniae isolates within a herd compared to isolates from different herds and this for both restriction enzymes used. Since the described PFGE technique was shown to be highly discriminative and reproducible, it will be a helpful tool to further elucidate the epidemiology of M. hyopneumoniae.     

Improved pulsed-field gel electrophoresis procedure for the analysis of Flavobacterium columnare isolates previously affected by DNA degradation

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host.     

Molecular genotyping of multinational ovine and caprine Corynebacterium pseudotuberculosis isolates using pulsed-field gel electrophoresis

Caseous lymphadenitis (CLA) is a chronic, suppurative disease, with a worldwide distribution, caused by Corynebacterium pseudotuberculosis. The clinical manifestation of CLA is known to vary between different countries, and has been postulated to be due to differences in the strains present in these countries. Forty-two sheep and goat isolates of C. pseudotuberculosis from Australia, Canada, Eire, The Netherlands and Northern Ireland were characterized by pulsed-field gel electrophoresis (PFGE), biotyping, antimicrobial susceptibility, and production of phospholipase D. The PFGE-determined genotypes of this multicentric collection were then compared with representative ovine and caprine isolates from a previously published panel of PFGE profiles of United Kingdom isolates. Digestion with SfiI generated 16-18 bands in the 48.5 and 290 kb range, and differentiated four distinct pulsotypes amongst the 36 ovine and 6 caprine strains which displayed remarkable homogeneity. Based on these results, it would appear that the genome of C. pseudotuberculosis is highly conserved, irrespective of the country of strain origin.     

环介导等温扩增联合横向流动试纸条快速检测单核细胞增生李斯特菌的研究

将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条检测技术(lateral flow dipstick,LFD)联合,建立了1种可应用于单核细胞增生李斯特菌快速检测的新方法。针对单核细胞增生李斯特菌的actA基因设计3对引物(其中,上游内引物由生物素标记)和1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针,进行由生物素标记的LAMP扩增反应,并利用LFD对经FITC标记探针杂交的扩增产物完成检测。优化后的LAMP反应条件为65℃反应40min,从细菌基因组DNA提取到LFD结果判断只需90min左右,比常规PCR技术缩短近2h。LAMP-LFD可特异性地检出单核细胞增生李斯特菌,对哈维弧菌等常见弧菌及嗜水气单胞菌等的检测结果为阴性。灵敏性试验表明,LAMP-LFD针对病原纯培养物的检测灵敏度为3.2×101 CFU/mL或0.64CFU/反应,是LAMP检测的10倍,常规PCR检测的100倍;针对人工污染单核细胞增生李斯特菌的原料奶样品的检测灵敏度为1.6×102 CFU/mL或3.2CFU/反应。利用本方法可从采集样品中检测到单核细胞增生李斯特菌,检测结果与传统的细菌分离培养方法结果一致。试验表明,本研究建立的LAMP-LFD技术可特异、准确地应用于单核细胞增生李斯特菌检测,而且灵敏度高、操作简单、检测成本低,有望发展成为单核细胞增生李斯特菌快速检测的有效手段。